Leishmania parasites are important human pathogens that cause a variety of disease ranging from cutaneous to visceral infections. Recently, the Leishmania Genome Network initiated a coordinated effort to sequence the Leishmania genome. As part of this effort, the smallest chromosome, 1 was recently sequenced. The genome project has generated a valuable resource of data and materials that will lay the foundation for additional cellular and molecular biology studies. We propose to utilize these tools to gain a better understanding of a fundamental cellular process, DNA replication, in Leishmania. The specific aims of this proposal are to identify the location and sequence of DNA replication origins; characterize the function of the replication origin sequences; and characterize chromosomal structure at the replication origins. DNA replication origins from 1 will be identified by 2D gel electrophoresis techniques and nascent strand analysis. The ability of these sequences to functionally initiate DNA replication will be assessed by gene targeting strategies including independently knocking-out and relocating these sequences in the chromosome. Finally, nuclease sensitivity will be employed to assess the local chromosomal structure and the accessibility of these sequences to the DNA replication machinery. The project is designed to generate valuable tools that could be exploited in future studies to advance functional studies of chromosomal structure, to characterize the proteins involved in the regulation of DNA replication, as well as to answer basic questions about DNA replication in Leishmania, all of which could lead to novel anti-parasitic therapies.